Already in 1873 Ernst Abbe found that beams of light coming from a light source are not focused by a lens into a zero-dimensional geometric focal point but, as a result of diffraction, only into a focal spot or focal volume extending around the geometric focal point. Normally diffraction leaves an elongated focal spot that is football-shaped. The minimum dimensions of this focal area are about Lambda/(2n), Lambda presenting the wavelength of the light, and n presenting the index of refraction. Along the optical axis the extension of the focal spot is at best only 4 times larger, namely 2×Lambda/(n). This barrier has various implications in many areas of technology. In fact, this applies to every application in which light is to be concentrated into a spatially limited area without contact. Examples encompass light microscopy, lithography, and the writing into optical data storage media.
In the field of fluorescence microscopy it is known from EP 0 801 759 B1 how to effectively reduce the area in which a sample is excited for fluorescence light emission to be specifically detected in a detector. According to that document the focal area of an excitation light beam is partially superimposed with the focal area of stimulation light beams which induce stimulated emission of the sample, by which the excited energy state of the sample out of which the spontaneous emission of fluorescence light takes place is de-excited again. Separation of the spontaneously emitted fluorescence light of interest from the light caused by the stimulated emission can be ensured by a disparity in wavelengths or by detecting the emitted fluorescence light at a different point in time. The spontaneously emitted fluorescence light, which is captured from the effectively reduced focal area of the excitation light beam, comes out of an area or volume which is smaller than the actual main focal area or volume of the excitation light beam.
A further starting point for reducing the effective focal area of a light beam is to produce an interference pattern in the focal area; to this end the excitation light beam is split up into partial beams, and the partial beams are superimposed with each other in a common focal area out of different directions so that the partial beams are preferably counter-propagating. The dimensions of the intensity maximum of this interference pattern now have a smaller extension of about Lambda/4n along the axis of counter-propagation. Located around the common geometric focal point, this main intensity maximum unfortunately is accompanied by at least two further secondary intensity maxima, situated in front of and behind the focal main maximum, but still within the common focal volume of the two partial beams.
From Stefan W. Hell “Increasing the Resolution of Far-Field Fluorescence Light Microscopy by Point-Spread-Function Engineering” in “Topics in Fluorescence Spectroscopy”; Volume 5: “Nonlinear and Two-Photon-Induced Fluorescence”, edited by J. Lakowicz, Plenum Press, New York, 1997, page 417 following, a method is known to erase these secondary maxima. This method relies on superimposing the interference pattern of the partial beams of the excitation light beams with another interference pattern of partial beams of a stimulating light beam causing stimulated emission, the interference pattern of the stimulation light beam having a minimum at the focus point, i.e. featuring destructive interference at the geometric focal point, and the wavelength of the stimulation light beam being twice that of the wavelength of the excitation light beam. In this way, the maxima of the stimulation light beam located in front of and behind the focal point overlap with the secondary intensity maxima of the excitation light beam, so that only the main maximum of the excitation light beam around the focal point is effectively excited for spontaneous emission of fluorescence light which is detected. In this prior art, the limitation to those cases in which the stimulation light has twice the wavelength of the excitation light is a serious drawback. Besides, an apparatus for the realization of this method requires extremely high alignment efforts since both the excitation light beam and the stimulation light beam have to be split up into partial beams and to be focused out of opposite directions into the same focal area. Moreover, the phase differences of both pairs of partial beams have to be simultaneously adjusted and controlled with regard to the kind of interference at the common focal point of the partial beams. While the excitation partial beam pair has to be brought to constructive interference, the stimulating partial beam pair has to be brought to destructive interference at the same geometrical focal point. Thus, a corresponding apparatus has in fact up to now not been realized, although it should potentially enable to reduce the effective area of excitation of a sample by the excitation light beam far below the barrier of Lambda/2n.
The present invention is not limited to applications in fluorescence microscopes. Instead, it extends to all cases in which an optical transition may be excited by excitation light, and in which the optical transition can somehow be influenced or counteracted by de-excitation light. This includes the case that an energy state is de-excited with the de-excitation light by means of stimulated emission. However, it is also included that the de-excitation light depletes a ground state which is the only state out of which the optical transition can be excited by the excitation light. Further, the optical transition to be excited may initiate a photo-chemical process which is somehow inhibited or at least hindered by the de-excitation light. Thus, the term de-excitation light and de-excitation light beam, respectively, do not have another meaning in the context of this description than that the optical transition to be excited is somehow influenced or counteracted. For the invention it is important to reduce the effective area of the excitation of the optical transition by means of the de-excitation light beam. This does, for example, not mean that the superposition of the de-excitation light beam with the excitation light beam requires simultaneous or synchronized occurrence in the focal area as long as the desired effect of the de-excitation light beam is still given within a sequence in time.